This project entitled "CFI Pathogen Inactivation Technology," is in response to the challenge of developing Enabling Technologies to "Ensure a safe and adequate blood supply through the development of new processing technologies" Topic 06-HL-106, p. 94 of the National Institutes of Health American Recovery and Reinvestment Act of 2009 Challenge Grant Omnibus Solicitation RFA-OD-09-003. The worldwide AIDS epidemic, the periodic emergence of Ebola and SARS, and the recent outbreaks of potentially pandemic strains of influenza such as H5N1 have highlighted a persistent concern in the healthcare community -- the need for effective pathogen inactivation and removal techniques for human blood plasma and plasma-derived products. There are also a number of emerging viruses such as West Nile and the breaking Mexican swine flu, and a number of potential bioterrorism pathogens such as smallpox that are of concern to the safety of the human plasma supply chain. In addition to viruses, bacteria and parasites such as Babesia spp. and Plasmodium spp. are major threats of spreading diseases through transfusion. The causes of the more rapid emergence and spread of these "killer" viruses and pathogens are not entirely known, but are thought to be caused by some combination of deforestation with urbanization of wild virus habitats, evolutionary mutations and rapid global travel. Annually, an estimated 3.8 million Americans are transfused with 28.2 million blood components derived from 12.8 million units of blood donated by apparently healthy volunteers. A rigorous scrutiny of blood donors and the screening of donated blood for various serological markers have significantly reduced the mortality and morbidity due to transfusion-associated infectious agents. Some enzyme immunoassays used for routine screening may detect viral antigens or antibodies, but not the infectious agents themselves. Thus, there could be an asymptomatic window period of infectivity responsible for a residual risk of post-transfusion infection. Current approaches such as pasteurization;solvent-detergent;UV irradiation;and chemical and photochemical inactivation not always effective against a wide spectrum of pathogens, are sometimes encumbered by process-specific deficiencies, and often result in denaturation of the biologics that they are designed to protect. We plan to develop a physical pathogen inactivation process for non-enveloped and enveloped viruses as well as pathogenic bacteria and parasites in human plasma and plasma protein products. The process utilizes supercritical and near-critical fluids (SuperFluids(tm) or SFS). SuperFluids(tm) are normally gases which, when compressed, exhibit enhanced solvation, penetration and expansion properties. These gases are used to permeate and inflate the virus and pathogen particles. The overfilled particles are then decompressed and, as a result of rapid phase conversion, rupture at their weakest points. Research to date indicates that the SuperFluids(tm) CFI (critical fluid inactivation) process inactivates both enveloped viruses such as MuLV, VSV, TGE, BDVD, Sindbis and HIV and nonenveloped viruses such as Polio, Adeno, Reo, Parvo and EMC while preserving biological activity of the treated product. In a research collaboration with the National Institute of Biological Standards and Control (NIBSC), London, England, we demonstrated that SuperFluids(tm) CFI can inactivate more than 4 logs of human Parvovirus B19 (one of the smallest and toughest viruses) in human plasma in a two-stage CFI unit in less than 20 seconds. We have also demonstrated that SFS can disrupt and inactivate microorganisms such as E. coli, and thick-walled prokaryotes such as B. subtilis and tough eukaryotes such as S. cerevisiae at viral inactivation SFS conditions. CFI can be used with conventional viral reduction methods such as SD and nanofiltration as an orthogonal method of pathogen clearance. Our specific plans for this challenge grant are to: (1) design and construct laboratory-scale prototypes to conduct evaluation and trade-off studies that will lead to the selection of a commercial-scale SFS-CFI design to achieve >6 logs of inactivation levels of nonenveloped and enveloped viruses with >90% retention of protein (e.g., Factor VIII) integrity;(2) test prototypical and emerging viruses and bacteria in human plasma in extant SFS-CFI units and in laboratory-scale prototypes in order to establish universal operation conditions as well as the universality of the SFS-CFI process for enveloped and nonenveloped viruses and pathogenic bacteria;(3) evaluate the potential of applying SFS-CFI enabling technology to human plasma proteins such as fibrinogen and other components;and (4) evaluate compatibility of SFS-CFI with other enabling pathogen inactivation/ reduction technologies such as nanofiltration to define orthogonal effective approaches to meet manufacturers'specifications and FDA requirements. Subsequently, with a pharmaceutical/biologics partner such as Baxter International and/or an institutional partner such as the American Red Cross, DOD or NHLBI, we plan to conduct pre-clinical studies, file an IND with the FDA and conduct clinical trials on CFI-treated plasma. A generally-applicable physical technology for inactivating viruses and emerging pathogens with high retention of biological activity will help ensure a blood supply that is safe from emerging and unknown pathogens as well as bioterrorism threats. In addition to human plasma and human plasma proteins, the developed technology will also be applicable to recombinant therapeutics, monoclonal antibodies, transgenics and vaccines. PUBLIC HEALTH RELEVANCE: There are a number of emerging viruses such as West Nile, Ebola, SARS, potential pandemic strains of influenza (H5N1), the breaking Mexican swine flu, bacteria, parasites and a number of potential bioterrorism pathogens such as smallpox that are of concern to the safety of the human plasma supply. Current approaches are not always effective against a wide spectrum of human and animal viruses, are sometimes encumbered by process-specific deficiencies, and often result in denaturation of the biologicals that they are designed to protect. CFI pathogen inactivation technology gives pathogens the "bends," inactivating them without damaging proteins and enzymes in medically important transfusion fluids such as human plasma. This purely physical technique does not involve the use of heat, chemicals and/or irradiation, each of which has significant drawbacks in the viral inactivation of human plasma. As such, while CFI is capable of inactivating wide classes of viruses, bacteria and parasites, CFI has negligible negative impact on biological integrity and potency of the treated fluids. We plan to develop this technology as an orthogonal virus inactivation technology to techniques such as solvent-detergent (SD) that is not effective against non-enveloped viruses and passive virus removal techniques such as nanofiltration which does not render viruses inactive. This orthogonal approach is consistent with the regulatory authorities in Europe and the US that require a minimum of two pathogen inactivation technologies, which work by different mechanisms of action. The potential impact of a generally-applicable physical technology for inactivating viruses and emerging pathogens with high retention of biological activity will be very significant. Such a technology, especially when used with conventional virus inactivation or removal methods such as SD or nanofiltration, will help ensure a blood supply that is safe from emerging and unknown pathogens and bioterrorism threats. In addition to human plasma and human plasma proteins such as fibrinogen, the developed technology will also be applicable to recombinant therapeutics, monoclonal antibodies, transgenics and vaccines.